Table 1.
Cell lines and clinical data.
Table 2.
Primers used for reverse transcriptase PCR.
Fig 1.
Effects of anticonvulsants on DNA synthesis of glioblastoma and brain metastasis low passage cell lines.
Glioblastoma cells (HROG02, HROG05, HROG15, HROG24) and brain metastasis cells (HROBL01, HROBML03 and HROBMC01) growing in 96-well half-area microplates were treated with (A, B) carbamazepine, (C, D) levetiracetam, (E, F) perampanel or (G, H) valproic acid as indicated for 48 h, before DNA synthesis was assessed with the BrdU incorporation assay. One hundred percent BrdU incorporation corresponds to cells cultured without anticonvulsant. Data are presented as mean ± SEM (n ≥ 12 separate cultures); *p<0.05 versus control cultures (Kruskal-Wallis test with post hoc Dunn’s test).
Fig 2.
Cell death, caspase activation, and FDG uptake.
(A) Subconfluent-growing glioblastoma cells were challenged with PER or solvent control DMSO, and caspase 3/7 enzyme activity was determined employing a colorimetric kit. Data are presented as mean ± SEM (n = 7–8 separate cultures). No significant caspase activation was observed (Kruskal-Wallis test with post hoc Dunn’s test). (B) Glioblastoma cells were exposed to highest dose of PER from caspase activation analysis for 48 h and of apoptotic (Sub-G1 peak) cells were estimated by cytofluorometric quantification. The portion of dead/apoptotic cells is expressed as percent of solvent treated control cultures. Data from n ≥ 5 separate cultures were used to calculate mean values ± SEM. No significant change in Sub-G1 fraction was observed (Mann-Whitney U test). (C) Glioblastoma cells were labelled with 18F-FDG, and tracer uptake was quantified. Counts per minute were normalized to the protein content of the samples. One hundred percent 18F-FDG uptake corresponds to solvent-treated tumor cells (n = 9; mean values ± SEM); *p<0.05 versus control cultures (Mann-Whitney U test).
Fig 3.
Glutamate release of glioblastoma and brain metastasis cells.
In subconfluent cell cultures, supernatants (w/o FCS) were collected for a total of 24 hours (± PER) and glutamate levels were determined. Extracellular glutamate levels were normalized to total protein levels of the cells. Data are presented as mean ± SEM (n≥14), *p<0.05 vs. solvent control (Mann-Whitney U test). Multiple comparisons versus control groups (two-way ANOVA with Bonferroni t-test) demonstrated an overall higher glutamate level in the supernatant of glioblastoma cells than in the supernatant of metastasis cells (p<0.001). Additionally, the two-way ANOVA also revealed a significant treatment effect, i.e. PER attenuated extracellular glutamate levels across all cell cohorts (p = 0.046).
Fig 4.
Effects of anticonvulsants on gene expression.
Both, glioblastoma and metastasis cells were seeded in 12-well plates. On the next day, medium was exchanged and the cells were exposed to carbamazepine, levetiracetam, perampanel or valproic acid at the indicated doses for 48 h. Subsequently, the mRNA expression of the indicated genes and house-keeping control GAPDH was analyzed by real-time PCR. (A) Relative amounts (2-ΔCt) of target mRNA of control cultures were compared for both cell entities (n ≥ 10 per cell line), *p<0.05 versus glioblastoma cells (Mann-Whitney U test). (B) Furthermore, effects of anticonvulsants were analyzed. Only genes with significant changes are shown in Fig 4B (n = 5–6 separate cultures per cell line; *p<0.05 versus control cultures (Kruskal-Wallis test with post hoc Dunn’s test)).